Biopharmaceutical Industry Practices for Sequence Variant Analyses of Recombinant Protein Therapeutics.

The application of advanced methodologies such as next-generation sequencing (NGS) and mass spectrometry (MS) to the characterization of cell lines and recombinant proteins has enabled the highly sensitive detection of sequence variants (SVs). However, although these approaches can be leveraged to provide deep insight into product microheterogeneity caused by SVs, they are not used in a standardized manner across the industry. Currently, there is little clarity and consensus on the utilization, timing, and significance of SV findings. This white paper addresses the current practices, logistics, and strategies for the analysis of SVs using a benchmarking survey coordinated by the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ) as well as a series of deliberations among a panel of experts assembled from across the biopharmaceutical industry. Discussion includes current industry experiences including approaches for detection and quantitation of SVs during cell-line and process development, risk assessments, and regulatory feedback. Although SVs are a potential issue for all recombinant protein therapeutics, the scope of this discussion will be limited to SVs produced in mammalian cells. Ultimately, it is our hope that the findings from the survey and deliberations of the committee are useful to decision makers in industry and positions them to respond to findings of SVs in recombinant proteins that are destined for clinical or commercial use in a strategic manner.LAY ABSTRACT: This white paper addresses the current practices, logistics, and strategies for the analysis of amino acid sequence variants using a benchmarking survey coordinated by the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ) as well as a series of deliberations among a panel of experts assembled from across the biopharmaceutical industry. Discussion includes current industry experiences regarding detection and quantitation of SVs during cell-line and process development, risk assessments, and regulatory feedback.

Implementation of Plate Imaging for Demonstration of Monoclonality in Biologics Manufacturing Development.

Monoclonality of mammalian cell lines used for production of biologics is a regulatory expectation and one of the attributes assessed as part of a larger process to ensure consistent quality of the biologic. Historically, monoclonality has been demonstrated through statistics generated from limiting dilution cloning or through verified flow cytometry methods. A variety of new technologies are now on the market with the potential to offer more efficient and robust approaches to generating and documenting a clonal cell line.Here we present an industry perspective on approaches for the application of imaging and integration of that information into a regulatory submission to support a monoclonality claim. These approaches represent the views of a consortium of companies within the BioPhorum Development Group and include case studies utilising imaging technology that apply scientifically sound approaches and efforts in demonstrating monoclonality. By highlighting both the utility of these alternative approaches and the advantages they bring over the traditional methods, as well as their adoption by industry leaders, we hope to encourage acceptance of their use within the biologics cell line development space and provide guidance for regulatory submission using these alternative approaches.LAY ABSTRACT: In the manufacture of biologics produced in mammalian cells, one recommendation by regulatory agencies to help ensure product consistency, safety, and efficacy is to produce the material from a monoclonal cell line derived from a single, progenitor cell. The process by which monoclonality is assured can be supplemented with single-well plate images of the progenitor cell. Here we highlight the utility of that imaging technology, describe approaches to verify the validity of those images, and discuss how to analyze that information to support a biologic filing application. This approach serves as an industry perspective to increased regulatory interest within the scope of monoclonality for mammalian cell culture-derived biologics.

Industrial production of clotting factors: Challenges of expression, and choice of host cells.

The development of recombinant forms of blood coagulation factors as safer alternatives to plasma derived factors marked a major advance in the treatment of common coagulation disorders. These are complex proteins, mostly enzymes or co-enzymes, involving multiple post-translational modifications, and therefore are difficult to express. This article reviews the nature of the expression challenges for the industrial production of these factors, vis-à-vis the translational and post-translational bottlenecks, as well as the choice of host cell lines for high-fidelity production. For achieving high productivities of vitamin K dependent proteins, which include factors II (prothrombin), VII, IX and X, and protein C, host cell limitation of γ-glutamyl carboxylation is a major bottleneck. Despite progress in addressing this, involvement of yet unidentified protein(s) impedes a complete cell engineering solution. Human factor VIII expresses at very low levels due to limitations at several steps in the protein secretion pathway. Protein and cell engineering, vector improvement and alternate host cells promise improvement in the productivity. Production of Von Willebrand factor is constrained by its large size, complex structure, and the need for extensive glycosylation and disulfide-bonded oligomerization. All the licensed therapeutic factors are produced in CHO, BHK or HEK293 cells. While HEK293 is a recent adoption, BHK cells appear to be disfavored.